Millions of people living with HIV (PLWH) will soon receive the integrase strand-transfer inhibitor (INSTI) dolutegravir (DTG), as the World Health Organization (WHO) now recommends DTG combined with two nucleoside reverse transcriptase inhibitors (NRTIs) as first- and second-line therapy. This combination therapy demonstrated very high efficacy in clinical trials and acquired drug resistance was absent (first-line treatment) or very rare (second-line treatment). It remains to be determined whether this high efficacy and lack of resistance development can be extrapolated to treatment of non-B subtype viruses in low- and middle-income countries where most PLWH live. Our long-term goal is to provide a scientific basis for optimal large-scale and long-term treatment of PLWH with DTG. Our central hypothesis is that novel viral mutations in and outside the integrase gene (with an emphasis on 3?-polypurine tract changes) contribute to virologic failure with DTG in clinical practice. This hypothesis will be tested by identifying PLWH failing DTG containing regimens in Brazil which is a model country for large-scale DTG implementation where various HIV subtypes co-circulate. Subsequently we will perform in-depth in vitro characterizations of novel viral mutations in and outside integrase gene in an HIV subtype comparative manner. The specific aims of this proposal are: 1) To characterize the pattern of INSTI resistance mutations that emerge during failure of DTG-containing cART in individuals infected with subtypes C, F, and BF recombinant forms in Brazil. These are the most prevalent non-B subtypes on the South-American continent. 2) To determine which changes in the 3?-PPT are observed in viruses from DTG failing individuals. Results of this study will show if this novel resistance pathway contributes to acquired drug resistance in clinical practice. 3) To determine the phenotypic consequences of mutations identified in specific aims 1 and 2. These studies assess and compare the effect of novel viral mutations on INSTI susceptibility and viral fitness in HIV subtypes A, B, C, and F in vitro (representing 70% of all infections worldwide), and the molecular mechanism of 3?-PPT mediated INSTI resistance will be determined. The insights generated with these studies will contribute to a more effective use of second generation INSTIs in the future.